Integrated affinity microcolumns and affinity capillary electrophoresis

ABSTRACT

Device and method for detecting the presence of known or unknown toxic agents in a fluid sample. Targets in the sample are bound to releasable receptors immobilized in a reaction region of a micro- or nano-fluidic device. The receptors are selected based on their affinity for classes of known toxic agents. The receptors are freed and the bound and unbound receptors separated based on differential electrokinetic mobilities while they travel to a detection device.

PRIORITY CLAIM

The present invention claims priority to U.S. Provisional PatentApplication No. 60/973,712, filed Sep. 19, 2007, the entirety of whichis hereby incorporated by reference.

FEDERALLY SPONSORED RESEARCH

The present invention was made with government support under Grant No.IIS0515684 awarded by the Defense Intelligence Agency through an NSFgrant. As a result, the government has certain rights in the invention.

BACKGROUND AND SUMMARY

It is widely acknowledged that there has been an increased threat ofchemical and biological weapons (CBWs). Recent events have made it clearthat CBWs pose a potential threat not only on the battlefield, but alsoas agents of terrorism. The agents under consideration range from lowmolecular weight compounds such as organophosphorus nerve agents toinvasive cells and viruses. In addition, many of these agents arealready established public health problems. See, e.g. Paddle B M. 2003.Therapy and prophylaxis of inhaled biological toxins. Journal of AppliedToxicology 23: 139-70, which is incorporated herein by reference.

Accordingly, detection of CBW agents is a continuing and acceleratingintelligence challenge. Detection of CBW agents is an exceptionallydemanding problem because the amounts of CBW agent sufficient to causeharm to humans is typically very small, requiring exceptionalsensitivity. Moreover, rapid identification and remediation isfrequently necessary. Even more worrisome, with advances in biologicalsynthesis capabilities, creation of new CBW agents is no longerexclusively a nation-state enterprise with large-scales observables, butis becoming a garage enterprise—on the scale of methamphetaminelabs—with widespread availability to potential adversaries.

Most current threat detection systems utilize immunology, PCR, orspectroscopic detection-based technologies which rely on preciseidentification of the biological or chemical toxin involved. While thisapproach has its uses, it is ineffective against either newly developedor modified threats that, by novelty or design, can evade preciserecognition elements.

Accordingly, there is a need for widely dispersible, inexpensive sensorsthat are able to monitor large areas for a wide variety of both knownand unknown agents. Accordingly, a chip-scale technology that issensitive to a variety of agent classes and that requires only verysmall sample volumes is needed.

Accordingly, in one embodiment, the present disclosure provides amicroscale, multi-threat agent detection system that is able to detectboth known and unknown agents by detection of physiological responsesassociated with exposure to a toxic agent, rather than the presence ofspecific toxins. In this strategy, the potential physiological effect iskey, and the exact identify of the threat agent is secondary. Detectionof physiological responses allows for rapid intervention and/orprophylaxis to block mortality and morbidity among potential targetpopulations. Because the detector exploits the target of the threat, orone of the targets of the threat, either novel threats, or thosedeliberately designed to thwart current detections schemes, are quicklydetected.

Moreover, at least in some embodiments, the system described herein,allows at least low level quantification of the ability of the threat tobind to the target physiological molecule, thus allowing for proper (orat least improved estimates of the proper) dosage of counter actingagents.

It will be appreciated that the need for such systems is apparent for avariety of applications, not limited to simply detection of CBW agents,but also including intelligence gathering, battlefield readiness,general public health, and both clinical and basic research.Accordingly, in at least some embodiments, the system described hereinis envisioned as an important component for future medical diagnosticand drug discovery applications, as well as being a possible means ofrapid and efficient proteomic analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic illustration of an exemplary detector accordingto a first embodiment.

FIG. 1B is a close up view of section of FIG. 1A.

FIG. 2 is a schematic illustration of an exemplary detector according toa second embodiment.

FIG. 3 is a schematic illustration of an exemplary detector according toa third embodiment.

FIG. 4 is a schematic illustration of an exemplary detector according toa fourth embodiment.

FIG. 5 is a schematic illustration of a method according to anembodiment employing the use of receptors encased in a lipid bilayerformed around a bead.

FIG. 6 is a schematic illustration of a method employing receptorsreversibly absorbed in a smart surface.

FIG. 7 is a schematic illustration of an exemplary detector according toa fifth embodiment.

FIG. 8 is a schematic illustration of an exemplary detector according toa sixth embodiment.

FIG. 9 shows a PDFMS microchannel with packed glass beads according tothe first Example.

FIG. 10 shows a confocal microscopy image of 30 μm glass beads withC₅-ganglioside G_(M1) receptor labeled with BODIPY dye packed in thePDMS microchannel of FIG. 8.

FIG. 11 shows voltages applied to different wells during sampleinjection.

FIG. 12 shows voltages applied to different wells during release andseparation.

FIG. 13 shows separation of receptor and receptor-ligand complex (4 μMCholera Toxin Subunit B).

FIG. 14 shows the binding curve for Cholera Toxin Subunit B andC₅-ganglioside G_(M1) receptor.

FIG. 15 provides fluorescence images of GM1 bearing beads packed in themicrochannel. Red signal is from cholera toxin B conjugated with thefluorophore, Alexa 555 and green signal is from GM1 receptor conjugatedwith the fluorophore, Bodipy FL. (a) Before injection of cholera toxin Bsample; (b) after injection of 100 nM cholera toxin B and washing; (c)after electrokinetic elution of GM1 and cholera toxin B with 10 wt %sodium dodecyl sulfate.

FIG. 16 shows the electrokinetic elution of GM1-cholera toxin B using 10wt % sodium dodecyl sulfate. The green line (below) represents theelution of GM1, while the red line represents the elution of choleratoxin B.

FIG. 17 depicts the elution of GM1 from microcolumns exposed toinjections (10 μL) of cholera toxin at various concentrations. 10 wt %sodium dodecyl sulfate was used for elution.

FIG. 18 is a dose-response curve for detection of cholera-toxin bymicrocolumn capture and capillary electrokinetic elution. Samplevolume=10 μL.

FIG. 19 depicts the elution of GM1 after exposure to duck pond waterspiked with 100 nM cholera toxin B. Sample vol.=10 μL.

DETAILED DESCRIPTION

Various embodiments of the present disclosure are configured to identifythe presence of a target using toxin receptor binding and electrokineticseparation. In general, the present disclosure provides a detectionsystem wherein a fluid sample is injected into the detector and allowedto interact with receptors reversibly immobilized in a reaction region.The receptors comprise known receptors that have been identified asbeing receptors for known targets or classes of targets (e.g. threatagents). It will be appreciated that according to various embodiments,the receptors may take the form of naturally-occurring or synthesizedversions of known receptors, naturally-occurring or synthesized versionsof modified known receptors, and/or naturally-occurring or synthesizedversions of biomimetics of known receptors. The receptors may befluorescently or otherwise labeled. Alternatively, because the presentlydisclosed methods rely on different mobilities for the receptor-ligandcomplex and the receptor, methods that utilize detection methods that donot require a label could be employed. Examples of suitable detectionsystems that do not necessarily require a label include, but are notlimited to electrical, acoustical, or UV detectors. If present, thetarget binds the receptors in the reaction region. Conditions are thenaltered such that the immobilized receptors are released from thereaction region, allowing both bound and unbound receptors to travelthrough a micro- or nano-fluidic channel in the detector to a detectionregion. As the receptors traverse the micro- or nano-fluidic channel,the bound and unbound receptors are separated in time based on thedifferent electrokinetic mobilities of the bound and unbound receptors.The passage of the receptors through the detection region is thenmonitored (e.g. by detecting the presence of a fluorescent label on thereceptors) and based on when signal is detected, a determination can bemade as to whether the sample passing through the detection regionincluded only unbound receptors (and therefore no target) or boundreceptors (therefore determining that the sample tested positive forpresence of the target).

Turning to FIG. 1A, a schematic illustration of an exemplary detector 10according to a first embodiment is shown. A silicon chip 12 includes twofluid injection columns 14 and 28 connecting a microfluidic channel 16.Columns 14 and 28 are in fluidic communication with microfluidic channel16 so as to allow for the introduction the various buffers, regents, andsample(s) into microfluidic channel 16. Microfluidic channel 16 includesone or more reaction regions 18, (shown in FIG. 1 as reaction regions 18a, 18 b and 18 c) and a detection region 26. A close up of a reactionregion 18 a is shown in FIG. 1B.

Each reaction region includes a target concentrating mechanismcomprising releasably immobilized receptors known to bind one or moreclasses of target agents. In the depicted embodiment, the targetconcentrating mechanism comprises biologically active beads 22. Forpurposes of the present description, the term “biologically activebeads” is intended to describe beads that are capable of interactingwith one or more target biological agents that may or may not be presentin a sample solution introduced into the detection device. For example,the surface of the beads may present one or more target-specificreceptors. More specific examples of biologically active beads will bedescribed below. In general, the receptors are associated with thetarget concentrating mechanism in such a way that they are immobilizedby the target concentrating mechanism under a first set of conditionsand are released by the target concentrating mechanism under a secondset of conditions. For the purposes of the present disclosure when areceptor is referred to as being “immobilized” by the targetconcentrating mechanism in the reaction region it is meant that thereceptor is prevented from leaving the confines of the reaction region.Several non-limiting mechanisms for immobilization are described ingreater detail below. Accordingly, when a receptor is “released” by thetarget concentrating mechanism, it is meant that the receptor, and anytarget to which the receptor is bound, is able to travel away from thereaction region.

Microfluidic channel 16 is further in communication with a fluidmanipulation source, which is capable of controlling fluid flow in oneor more desired directions. Accordingly, a sample fluid is introduced incolumn 14 and encouraged to flow in the x direction, shown by the arrowtowards the reaction and detection regions 18 and 26. Suitable fluidmanipulation sources include external hydraulic pumps, electrodesconfigured to induce electro-osmotic pumping, pressure-driven flow,combinations thereof, and the like.

Depending on the desired detection profile of the detector, reactionregions 18 a, 18 b, and 18 c may include the same or different receptorsconfigured to bind the same or different targets. For example, a firstreaction region could include receptors known to bind a first class ofthreat agents or associated with a first physiological effect, while asecond reaction region could include receptors known to bind a secondclass of threat agents or associated with a second physiological threat.Of course it will be understood that while three reaction regions areshown for illustrative purposes, more or fewer reaction regions could beutilized depending on the desired detection profile.

FIG. 2 depicts another embodiment of a detector 10 b according to thepresent disclosure. In this embodiment, the microfluidic channelincludes a plurality of detection regions, 26 a, 26 b, and 26 c. As withthe embodiment shown in FIG. 1, reaction regions 18 a, 18 b, and 18 cmay include the same or different receptors configured to bind the sameor different targets. Furthermore, the mechanism(s) used for detectionin detection regions 26 a, 26 b, and 26 c may be the same or differentin each region. Again it will be understood that the number of detectionand reaction regions shown is for illustrative purposes only and shouldnot be considered in a limiting fashion.

FIG. 3 depicts yet another embodiment of a detector 10 c according thepresent disclosure. In this embodiment, the chip 30 includes twomicrofluidic channels 40 and 42 in a t-pattern. The terminal end of eachchannel is in fluidic communication with a column: identified as columns32, 34, 36, and 38, respectively. The long end of the “t” shape includesone or more reaction regions 18 and one or more detection regions 26. Itwill be appreciated that the configurations of either FIG. 1 or FIG. 2may be employed or that alternative, suitable configurations for thereaction and detection regions may be employed. In this embodiment,fluid manipulation source are used to control fluidic movement in the xand y directions. In practicality, if this geometry is used, it may benecessary to employ three fluid manipulation sources, one to encouragemovement in the y direction during the reaction phase, one to encouragemovement in the x direction during the reaction phase, and one todiscourage movement in the y direction during the detection phase. Inthis embodiment, a fluid sample is introduced into channel 34 and thenmoved along channel 42 (e.g. in the y direction) towards the reactionchamber 18 at the intersection with channel 40. Reversibly immobilizedreceptors inside of reaction chamber 18 bind the target, while waste isallowed to continue down channel 42 (continuing in the y-direction)towards column 36. The immobilized receptors are then released from bythe target concentration mechanism (as described in greater detailbelow) and encouraged to flow down microcolumn (or “detection lane”) 40(in the x direction) towards detection region 26.

Turning now to FIG. 4, it can be seen that the geometry shown in FIG. 3can be expanded to provide multiple detection lanes, thereby providing atwo-dimensional arrayed detector 10 d. In this example, a sample isintroduced to column 50 and encouraged to flow through microchannel 52through a series of reaction regions 18 a-18 e. Each reaction regionmay, for example, include receptors selected to interact with the sameor a different target or a different class or type of target. Conditionsare selected such that any target present within the sample is able tobind suitable receptors with the desired degree of specificity. Then,conditions are altered, as described in greater detail below, such thatthe target concentrating mechanism releases the receptors. The bound andunbound receptors are then encouraged to flow down an associatedmicrochannel 54 a-54 e, (i.e. microchannel 54 a for reaction region 18a) towards the corresponding detection region 26 a-26 e (i.e. detectionregion 26 a for reaction region 18 a). Conditions are provided such thatduring the travel from the reaction region to the detection region, thebound and unbound receptors separate from each other and the timing ofthe passage of the receptors through the detection region is monitored.

It should be appreciated that any geometry may be employed in thepresently-described detection system. For example, in some cases thegeometry may, at least in part, be determined by the differentmobilities of the receptor and receptor-ligand complex. As the mobilitesof the receptor and receptor-ligand complex are more similar, a longerseparation pathway may be required in order to attain detectableseparation. Accordingly, non-linear geometries including U-shapes,spirals, or the like may be employed in order to attain the desiredseparation while confining the system to a given space. Furthermore, insome cases it may be possible or even desirable to use 3-dimensionalgeometries. Moreover, it will be appreciated that the mechanism(s) usedto encourage fluid flow in the system may be affected and/or determinedby the particular geometry used. Accordingly, 3-dimensional (or some2-dimensional) geometries may employ gravity-driven, magnetically-drivenor cryogenically-driven fluid flow systems in additional to or as analternative to the various fluid-flow mechanisms identified above.

It will be appreciated that the microchannels described herein may beformed using any suitable method including by employing standardphotolithography techniques. An example of a useful technique forfabricating the chips herein is described in O'Brien et al. 2003“Fabrication of an integrated nanochip using interferometriclithography.” Journal of Vacuum Science and Technology B 21: 2941-5,which is hereby incorporated by reference. Generally, interferometriclithography (IL) and lift off are used to form a nanopatterned hardmetal (e.g., mask) over the entire surface of a silicon wafer (e.g.,2″). Conventional projection lithography techniques are then used todelineate with photoresist the areas corresponding to the microfluidic(e.g., 200 μm wide) connections, the microscale grating structureswithin the microfluidic channels (which may be used to trap the beads inthe reaction region(s) and shown, for example, in FIG. 1B at 24), themacroscale (2 mm dia.) reservoirs at the ends of the microfluidicstreams, and the entire nanochannel array (analysis stream). Thepatterned (IL hard mask and conventional photoresist) chip is thensubjected to reactive ion etching to form the three dimensional reliefof the analytical chip. Upon removal of the photoresist and the hardmask, cleaning, and oxidation as desired, the entire fluidic system isenclosed by anodic bonding of a transparent solid (such as Pyrex®glassware available from World Kitchen, LLC) “roof” (that contains theinterface ports) over the entire surface of the patterned chip.Additional macroscopic connectors can be glued or otherwise attached tothe top of the chip to act as additional fluid reservoirs or asinterfaces to macroscale pumping systems. Those of skill in the art willbe familiar with a variety of methods and mechanisms that are useful forthe formation and use of microfluidic devices and such methods andmechanisms (i.e. sample injectors, valves, pumps, mixers, bead-packedmicrocolumns) may be incorporated herein, as desired or necessitated bythe specific design. Exemplary methods and mechanisms are described, forexample, in Piyasena et al., 2004 “Near-simultaneous and real-timedetection of multiple analytes in affinity microcolumns.” AnalyticalChemistry 76: 6366-273 and Piyasena et al., 2006 “An electrokinetic cellmodel for analysis and optimization of electroosmotic microfluidicpumps.” Sensors and Actuators B 113: 461-467, each of which is herebyincorporated by reference.

To prepare the chip for experimentation, it may first be loaded withaqueous buffer solution via capillary action. In the t-shapedgeometries, the detection microchannel (i.e. microchannels 40 and 54 inFIGS. 3 and 4, respectively) is typically filled first until the entirearray is saturated. At this point, the remainder of the chip can befilled by introducing buffer through the sample stream leg. Once theentire chip is filled, fluid flows in each leg of the system through thefluid manipulation source.

Regardless of the particular geometry used to configure themicrochannels, reactions regions and detection regions, as stated above,each reaction region includes a target concentrating mechanism whichmay, for example, take the form of a plurality of biologically activebeads. Formation of packed bead microcolumns may be accomplished by theintroduction of biologically-active beads through a bead packing stream.Grates in the sample and waste streams can be constructed as describedabove in order to sequester the beads in one or more desired locations(e.g. the reaction regions). For example, in the device shown in FIG. 3,it may be desirable to sequester the beads in the junction of lanes 40and 42. In some embodiments, a physical occlusion in the bead packingstream may not be necessary to maintain the packing because fluid flowcan be controlled using the fluid manipulation source. Alternatively,beads may be packed sequentially such that a frit can be formed fromreactive beads after the biologically active beads are introduced. Seee.g., Piyasena M E et al., 2004 “Near-simultaneous and real-timedetection of multiple analytes in affinity microcolumns.” AnalyticalChemistry 76: 6366-273, which is hereby incorporated by reference.

As stated above, the regardless of the geometry used, thepresently-described detectors all employ a target concentrationmechanism within the reaction region that is configured to reversiblyimmobilize receptors within the reaction region. All threat agents,regardless of their source, exert their toxicity at the cellular level,which requires interaction with the cell or cellular triggers. Theinteraction is often mediated by binding of or interaction between thethreat agent and a specific receptor, (or binding partner). For example,nerve agents bind to receptors for neurotransmitters, and Shiga andCholera Toxins bind glycosides on ion channels. Thus, the agentstypically must be able to bind specific receptors in order to affecttheir desired physiological effect. Accordingly, even if a threat agenthas been modified (either through natural mutation or in a laboratory),it will typically retain or include a binding region associated with atarget receptor. It is this biological mechanism that is exploited bythe presently-described system so as to be able to detect both known andunknown threat agents.

There are a number of biological receptors that are known to be strongtargets for CBW agents because binding of these targets by a toxinproduces known physiological effects. Consequently, molecules suitablefor use in the detection system described herein include specificbiomolecules targeted by known classes of threat agents. For example, itis well known that nerve agents (e.g. soman, sarin) interact with thesoluble enzyme acetylcholine esterase (AChE). For that reason, AchE maybe a suitable receptor for the methodologies described herein. Inaddition, these agents are known to bind to muscarinic receptors,further amplifying the results of an increased amount of acetylcholineon the parasympathetic nervous system. Thus, solubilzed muscarinicreceptors may similarly be a suitable receptor for use with themethodologies described here.

Another class of nerve agents is reflected in the shellfish paralysisagents (SPAs), also known as paralytic shellfish toxins (PSTs), whichblock ion channels. Dinoflagellates, (the cause of “red tide”) producetoxins which are accumulated in filter feeders such as shellfish.Similar compounds are found in the toxic organs of puffer fish. Thesetoxins are related to the archetypal molecule saxotoxin and itsanalogues. A naturally occurring compound, saxiphilin has been found inthe blood of many marine invertebrates. Saxiphilin is a known receptorfor SPAs, the function of which is to sequester SPA-type compounds.Accordingly, the hydrophilic protein receptor saxiphilin may also besuitable for use with the methodologies described here.

Bacterial agents exhibit their pathology through specific toxins.Specific cellular targets for many threats such as anthrax have beenidentified. See, e.g., Bradley K A, et al., 2003. “Anthrax toxinreceptor proteins” Biochemical Pharmacology 65: 309-14, which is herebyincorporated by reference. In addition, generalized targets, such asT-cell receptors, which react to a variety of pathogenic bacteria,ranging from relatively benign infections with Pseudomonas aeruginosa,to plague and tularemia, have been discovered. See e.g. Gossman et al.,2002. “Quantitative structure-activity relations for γδT cell activationby phosphoantigens.” Journal of Medicinal Chemistry 45: 4868-74, whichis hereby incorporated by reference.

Enterotoxins represent an important general class of bacterial toxins.These food or water born toxins cause severe, hemorrhagic diarrhea oftenleading to death. They usually consist of two subunits: one that bindsto receptors on the intestinal mucosa, and the other which permeates thecell membrane. The non-toxic subunits of these could be used fordetection. Since many of these toxins share similar binding sites (e.g.shiga, cholera and enteropathic E. coli), known receptors could be usedto screen for other toxins of this class.

Viral agents, although not currently weaponized, could, in fact, lead toa new generation of bioterror. Among the possible threats includehantaviruses, filoviruses (e.g. Ebola), and variations of pox viruses.See e.g. Su JR. 2004, “Emerging Viral Infections” Clinical andLaboratory Medicine 24: 773-95, hereby incorporated by reference.Viruses require entry into cells for propagation and the first step incellular infection is binding to a cellular receptor. For hantaviruses,for example, β-3 integrins, present on endothelial cells, seem to be themajor target. Ebola and other lentoviruses seem to enter throughdendritic receptors. See e.g. Watson, et al., 2002. “Targetedtransduction patterns in the mouse brain by lentivirus vectorspseudotyped with VSV, Ebola, Mokola, LCMV, or MuLV envelope proteins.”Molecular Therapy 5: 528-37, hereby incorporated by reference.

Some of the most potent potential toxins, e.g. aflatoxin, exert theireffects on the cell in part by intercalating into DNA. Therefore,detection of these agents might be easily accomplished using doublestranded DNA as receptor.

Accordingly, it can be seen that there are a large number of knownreceptors that are suitable for use with the present disclosure.

As stated above, in some embodiments, the target concentration mechanismtakes the form of a biologically active bead. According to one specificembodiment, the desired receptor (or receptors) are encased in a lipidbilayer formed around a silica (or other suitable) bead. As shown inFIG. 5, the biologically active bead includes the silica bead 100, thelipid bilayer 102 and the receptor 104. It is this biologically activebead that is then packed into the reaction region of the detector. Asample suspected of including a toxin analyte 106 is introduced (forexample as described above) under suitable conditions to allow forbinding, producing a bead having a receptor-ligand complex 108 boundthereto. Surfactant is then introduced to disrupt the lipid bilayeraround the bead and release the bound receptor-ligand complex resultingin the naked silica bead 100, unbound receptor 104 (which may or may notbe present, depending on the amount of analyte initially present in thesample), excess toxin analyte 106 (which also may or may not be present,depending on the amount of analyte initially present in the sample), andthe released receptor-ligand complex 108. The unbound toxin analyte 106and bound receptor-ligand complex then travels to the detection region.

In general, the unbound toxin analyte, unbound receptor, and boundreceptor will have different electrokinetic mobility due to thedifference in their molecular weight and charge. Some embodiments maytake advantage of this difference by electrokinetically separating thedifferent molecules before detection. Accordingly, movement of theunbound toxin analyte, unbound receptor, and bound receptor may beaccomplished by electrophoresis. Alternatively, the detector may employother (or additional) mechanisms for separately detecting the unboundreceptor and the bound receptor. For example, the separation channel(i.e. the channel leading from the reaction region to the detectionregion) may employ physical or other modifications configured to allowthe bound and unbound receptor to be differentiated. For example,physical barriers may be present which slow the progress of the largerbound receptor-ligand complex relative to the smaller unbound receptor.Alternatively or additionally, the separation channel may by modifiedwith a hydrogel such as poly(ethylene glycol) (“PEG”), polyacrylic acidor polyacrylamide, and/or include gel monoliths formed frompolyacrylamide or the like, porous polymer monoliths, choromotographicpacking, patterned silica nanospheres, etc. Moreover, it will beunderstood that alternative, non-electrokinetic, separation methods suchas pressure driven separation may be employed.

In a still further embodiment, the target concentrating mechanismcomprises a stimuli-responsive polymer (SRP) (Also referred to herein asa “smart polymer” or “smart surface”). Stimuli-responsive polymers aredescribed, for example, in U.S. Pat. Nos. 6,491,061, 6,615,855, and6,755,621, and U.S. patent application Ser. No. 11/682,396, each ofwhich is hereby incorporated by reference. See also, Fu et al., 2003“Control of molecular transport through stimuli-responsive orderedmesoporous materials.” Advanced Materials 15:1262-6; Ista et al., 2001“Synthesis of poly(N-isopropylacrylamide) on initiator-modifiedself-assembled monolayers. Langmuir 17:2552-5; Ista et al., 1999“Surface-grafted, environmentally responsive polymers for biofilmrelease.” Appl Environ. Microbiol. 65: 1603-9; Balamurgan et al., 2003“Thermal response of poly(N-isopropylacrylamide) brushes probed bysurface Plasmon resonance.” Langmuir 19: 2545-9; and Filipcsei, et al.,2007 “Magnetic Field-Responsive Smart Polymer Composites.” Adv Polym Sci206:137-189, each of which is also incorporated by reference. Ingeneral, the term “stimuli-responsive polymer” refers to synthetic,naturally occurring and semi-synthetic polymers which exhibit rapid andreversible changes in conformation as a response to environmentalstimuli. Examples of environmental stimuli can include temperature, pH,ionic strength, electrical potential, light intensity and lightwavelength. As described in these references, the stimuli-responsivepolymer can be used to control molecular transport of aqueous solutes.According to one particularly described embodiment, a porous networkincluding SRPs enables dynamic control of size-selective transport.Accordingly, such a porous network could be used in the presentlydisclosed system as a mechanism to both concentrate and separate boundreceptor-ligand complexes from unbound receptors.

As described in the U.S. patent application Ser. No. 11/682,396, theporous network containing the SRPs make take the form of a bead (whichmay be referred to herein as a “smart bead.”) Those of skill in the artwill be familiar with methods for forming beads of mesoporous material.Exemplary methods are described in U.S. patent application Ser. No.10/640,249 and U.S. Provisional Patent Application Ser. No. 60/985,050,each of which is hereby incorporated by reference. See also, Rao et al.,2000 “Encapsulation of poly(N-isopropyl acrylamide) in silica: Astimuli-responsive hybrid material that incorporates molecularnano-valves.” Advanced Materials 12: 1692-5, which is also incorporatedby reference.

Turning now to FIG. 6, a smart bead 110 to which a plurality ofreceptors 104 have been reversibly absorbed is shown. The smart bead isthen exposed to a fluid sample suspected of containing an agent ofinterest 116. Any agent of interest present in the fluid sample bindsthe receptors. The smart beads are then exposed to appropriateenvironmental conditions to allow for release of the receptors from thesmart bead. Accordingly, both bound 118 and unbound 116 receptors arereleased into the fluid flow. In the example shown in FIG. 6, theunbound receptors reach the detection region 126 first and the boundreceptor-ligand complexes second.

It is noted that in FIG. 5, both the receptors and the agent of interestare shown bearing detectable labels 109 and 111, respectively and thatno labels are shown in the embodiment of FIG. 6. As described elsewherein the present disclosure, labels may or may not be used, depending onthe particular detection system employed. For the proof of conceptexperiments described in the Example sections below, both the receptorsand the agent were labeled, in order to demonstrate that detectableseparation of the bound and unbound receptor complexes was achieved.However, it will be understood that labeling of one or more of thevarious experimental components, while certainly possible, is notnecessitated by the methods described herein.

Accordingly, in one embodiment, the biologically active beads of thepresent disclosure are smart beads decorated with reversibly absorbedreceptors, such that the receptors can be released upon exposure of thesmart bead to the appropriate environmental stimulus. Methods fordecorating smart surfaces with reversibly absorbed receptors aredescribed in Balamurugan et al., 2005 “Reversible Protein Absoption andBioadhesion on Monolayers Terminated with Mixtures of Oligo(ethyleneglycol) and Methyl Groups” J. Am. Chem. Soc. 127: 14548-14549, which ishereby incorporated by reference. It will be appreciated, of course,that while the smart surface shown in FIG. 6 is in the shape of aspherical bead, any suitable surface shape or configuration may be usedincluding, but not limited to spherical, or non-spherical beads, planarconfigurations, matrices, etc.

As an exemplary mechanism and method, a microfluidic chip, as describedabove, may have one or more reaction regions comprising a plurality ofsmart beads including reversibly absorbed receptors that bind to one ormore classes of CBWs. The receptors may be fluorescently or otherwiselabeled, or not, as determined by the detection method being used. Thesample stream is then passed through the reaction region(s) such thatCBW agents, if present, bind to the immobilized receptors. Because theimmobilized receptors act to concentrate the threat agent on the beads,extremely sensitive detection is possible, even with arbitrary samplevolumes. In the case of the t-shaped configurations, the fluid flowdirectionality is altered once sampling is completed. Regardless of theconfiguration used, once sampling is completed, the smart beads areexposed to the appropriate environmental stimulus to effect release ofthe immobilized receptors. As in the embodiments described above, thebound and unbound receptors then flow through a microfluidic channel,where they are separated, and the timing of the passage of the receptorsthrough the detection region is then determined. As before, thedifferences in eletrokinetic mobilities between the unbound receptorsand bound receptor/agent complexes can be exploited to indicate thepresence, concentration, and possibly identity of CBW agents present inthe sample.

As stated above, in some embodiments the detection system is based onthe differential electrokinetic mobilities of bound and unboundreceptors within the microfluidic arrays. Specifically, it is expectedthat the bound and unbound receptors will separate into twotime-separated detectable clusters as they travel through themicrofluidic channel towards the detection region. The expected time forunbound receptors (t_(r)) can easily be determined by simply running theexperiment without target sample. Accordingly, if a signal is detectedat a time point that is statistically different from the expected t_(r)it can be determined that the sample contains the target agent.Moreover, detecting two time-separated signals may be enough todetermine that target is present in the sample.

In another embodiment, the device may include a control lane thatoperates under the same conditions and responds to the same fluidmanipulation source, but which is not exposed to target. Accordingly, bycomparing the time point of the signal detected in the control lane withthe time point(s) of signal(s) detected in the test lane(s), the usercould determine whether or not target is present in the sample. FIGS. 7and 8 depict two exemplary embodiments of devices including controllanes. In FIG. 7, the device 210 contains two separate lanes, controllane 212 and test lane 214. Control lanes 212 and 214 are both connectedto the same fluid manipulation source 216. Accordingly, the user couldinject a sample for detection into inlet 218 in lane 214 and an inertfluid, such as electrolytic solution into inlet 220, and encouragemovement of the fluid in the microfluidic device using the same fluidmanipulation source. In the embodiment depicted in FIG. 8, control lane222 and test lane 224 are in fluid communication with one another sothat identical reaction conditions are achieved in both lanes. However,a fluid manipulation source ensures that fluid does not flow from lane224 to 222, ensuring the control lane is not contaminated with targetfrom the sample. Those of skill in the art will contemplate that a widevariety of geometries and configurations including control lanes areavailable and that those provided here are provided only for purposes ofillustration.

It will be understood that in some embodiments it may be desirable todetermine not only whether or not a CBW threat is present, but toattempt to garner more specific information about the particular threatidentified in the sample. Accordingly, the principles of flow cytometryand Affinity Capillary Electrophoresis may be used to develop and builda database of expected ligand/receptor complex behaviors to serve as anaid in analysis of test results. In this embodiment, the targetconcentrating mechanism, whether in the form of a smart bead or not, maycomprise a precisely defined concentration of receptors in order toproduce consistent, repeatable results to allow for various analyses ofspecific previously-identified CBW agents and known potential threats.Flow cytometry, a method of obtaining precise fluorescence spectrometricdata from individual particles (e.g. cells or microbeads) is a veryuseful method for determining the concentration of receptors on thesurfaces of beads used to construct affinity microcolumns. See e.g.“Buranda et al., 2002 “Biomolecular recognition on well-characterizedbeads packed in microfluidic channels.” Analytical Chemistry 74:1149-56.

Moreover, flow cytometry can also be used to determine the affinity ofreceptors toward model CBW-relevant ligands as well as the rate ofdissociation of model ligand/receptor complexes. Such information can beused to develop and build the aforementioned database of expectedligand/receptor complex behaviors. For example, it will be understoodthat agents that bind the same receptor may demonstrate differentbehaviors during transportation (e.g. based on size, electrochemicalcomposition, or the like) and knowledge of such differences may allowthe user to more specifically identify the particular agent presentwithin the sample. As a specific example, such a database may be able toidentify the time that a particular agent would be expected to take totravel from the target concentration region to the detection regionafter release from the target concentration region (i.e. the “traveltime” for that agent). Since it would be expected that different threatsmight have different expected travel times, a user could detect not onlythe presence of the threat, but also the possible identity of the threatby determining the travel time.

The above-described embodiments have discussed the use of microfluidicchannels, which are generally described as channels having at least onedimension in the range of 1-100 microns. However, the present disclosurealso provides for the use of nanofluidic channels within the detectiondevice. For the purposes of the present disclosure, nanofluidic channelsthose channels which are identified as having at least one dimensionsmaller than one micron. Using fluid volumes in the nanoscale rangesignificantly reduces the size of the sample required.

It should be noted that in nanoscale channels, the electrostatic effectsof electro-osmotic flows and steric effects can have profound effects onanalyte separations. For example, when a sample including two separatefluorescent dyes—one negatively charged and one positively charged isinjected into a nanofluidic device such as that of the presentdisclosure, electrokinetic separation is faster than and in the oppositedirection from a similarly designed microfluidic device, that is, in thenanofluidic device the negatively charged dye travels to the cathodefaster than the neutral dye, while in the microfluidic device, theneutral dye travels to the cathode faster than the negatively chargeddye. This behavior was demonstrated in a chip using a T-shaped geometryincluding nanfluidic channels intergrated with microchannels with ahierarchical combination of pattern features ranging over a span of sixorders of magnitude—from ˜1-cm flow lengths to 50-nm nanfluidic channelwidths. See e.g. e O'Biren et al., 2003 “Fabrication of an integratednanochip using interferometric lithography.” Journal of Vacuum Scienceand Technology B 21: 2941-5. Initial demonstrations of molecular flowand separations in these nanochannels offer a unique experimentalplatform for nanofluidics because for the first time, the Debyescreening length is comparable to channel width. This anomalous behaviorresults from the enhanced importance of screening and fluid/channel-wallinteractions in these nanoscale channels. In other words, at thenanoscale, molecular and surface interactions dominate transport. Theelectrical double layers that arise in solutions of electrolytes due toscreening of surface charge at ionic surface are ˜10 s of nmwide—comparable to the channel width. The scale of these layers can becontrolled by external biasing (analogous to charge transport infield-effect transistors) creating an entirely new approach to fluidcontrol. See e.g. Garcia et al., 2005, “Electrokinetic molecularseparation in nanoscale fluidic channels.” Lap Chip 5, 1271-1276. Thesebehaviors can be studied and catalogued in order to allow for the moreprecise characterization of CBW threats in sample populations. Moreover,nanofluidic devices such as those described herein can be used as aninexpensive, facile and manufacturable means for creating integratedfluidic circuits that allow the transition from macroscopic fluidhandling (e.g. pipettes) to nanoscale dimensions.

Further understanding of the present disclosure may be had by review ofthe following examples:

Example I Differential Migration of Cholera Toxin Subunit B andC₅-ganglioside G_(M1) Receptor in T-Microchannel

Preparation of T-Microchannel

T-Microchannel was fabricated with polydimethylsiloxane (PDMS) polymerusing soft lithography method. PDMS microchannel was fabricated withthree weirs at T cross-section to hold 30 μm beads. The dimensions ofmicrochannel were: NS length 6 cm, WE length 3 cm, WC and EC length 1.5cm, NC length 1.0 cm, width 300 μm and height 100 μm.

Preparation of Microsphere Supported Lipid Bilayers Incorporated withReceptor Protein

1 mM solution of egg phosphatidylcholine (egg PC) in chloroform (200 μltotal volume) was taken in a clear glass tube. 10 μl (2.5 mg/ml) ofC₅-ganglioside G_(M1) receptor labeled with BODIPY dye was added to eggPC solution. Dry nitrogen gas was bubbled through the solution todryness, leaving a film at the bottom of the glass tube. The film wassubsequently vacuum dried at room temperature for half an hour. Afteraddition of 1 ml of Tris buffer (pH 8.3) the solution was sonicated tooptical clarity in a sonication bath. 30 μm glass beads were added tothe small unilamellar vesicles dispersions with vortexing for 2 minutesin a microfuge tube. In this manner small unilamellar vesiclesspontaneously collapsed into a continuous bilayer incorporated withreceptor protein surrounding beads. After sitting for 30 minutes, thebeads were then centrifuged and resuspended in buffer, repeating forfifteen times to remove unbound lipid and receptor protein. These glassbeads with lipid bilayers and receptor protein were then packed in PDMST-microchannel with vacuum. FIG. 10 shows a Confocal Microscopy image of30 μm glass beads with C₅-ganglioside G_(M1) receptor labeled withBODIPY dye packed in PDMS microchannel.

Binding, Release and Detection of Toxin Based on ElectrokineticSeparation

Cholera Toxin Subunit B was used as a ligand. FIG. 11 shows the voltagesapplied at different wells for electrokinetic sample injection. CholeraToxin Subunit B sample was added in E well. Sample was injected due toelectrokinetic mobility. Cholera Toxin Subunit B binded toC₅-ganglioside G_(M1) receptor on beads and formed the complex. After 20minutes of sample injection voltages were switched (FIG. 12) and at thesame time 10% sodiumdodecylsulfate (SDS) surfactant was added in N well.Due to SDS, receptor-ligand complex and unbound receptor from the beadsgot released and injected in separation channel (CS). Receptor-toxincomplex, unbound receptor and excess toxin eluted in separation channelwere detected by Confocal Microscopy at a distance of 7 mm from point 2.FIG. 13 shows the separation order for 4 μM Cholera Toxin.Receptor-toxin complex and receptor were separated and detected due todifference in the electrokinetic mobility. FIG. 14 shows the bindingcurve for Cholera Toxin Subunit B and C₅-ganglioside G_(M1) receptor.

Example II Rapid Prototyping of Microfluidic Chips with Bead-PackedAffinity Microcolumns

A reproducible protocol for rapid fabrication of polydimethylsiloxane(PDMS) microchannels via soft lithography and packing receptor-bearingaffinity beads into packed beds of controlled lengths is demonstrated.We have used two microfluidic configurations, straight channels andT-cross section chips. Soft lithography enables the facile redesign andprototyping of channel configurations and dimensions such that chip,microcolumn and analysis parameters can be optimized. Using thesetechniques, it is possible to generate large numbers of chips fortesting of toxin-detection performance under a variety of experimentalconditions. We have developed standard operating procedures formicrocolumn packing, sample introduction, pumping, analyte capture,analyte release, electrokinetic separation of receptor andreceptor/toxin complexes, and finally detection of receptor andreceptor/toxin complexes. A number of microfluidic separation matriceshave been explored thus far. The results shown below are obtained usingtraditional microchannel capillary electrophoresis. Our preliminaryresults suggest that much more efficient separations will be enabledthrough the integration and use of nanofluidic channel arrays.

Preconcentration of Toxin on Biomimetic Beads and Elution ofReceptor-Toxin from Microcolumns.

We have demonstrated preparation of biomimetic affinity beads with GM1as a receptor incorporated within EggPC lipid bilayers supported onsilica beads. Cholera toxin and other enterotoxins bind to the GM1receptor. The GM1/cholera toxin B pair is one of the best-studiedreceptor/toxin systems and thus this model receptor/toxin system is wellsuited for these studies. We have demonstrated the efficient binding ofcholera toxin B to GM1 supported on biomimetic beads packed inmicrofluidic channels. Preconcentration of the toxin was achieved usinga microcolumn packed with receptor bearing affinity beads. FIGS. 15A-15Cshow the fluorescence images of beads packed in PDMS straight channel.In order to demonstrate detectable separation of the unbound toxin,unbound receptor, and bound receptor-toxin complex, both the toxin andthe receptor were labeled in this experiment. Accordingly, the signalshown in the upper panels (which would appear red in a color image) isfrom cholera toxin B conjugated with ALEXA FLUOR 555® fluorophore andthe signal shown in the lower panels (which would appear green in acolor image) is from BODIPY FL® fluorophore conjugated to GM1 receptor.FIG. 15A shows 30 μm beads before cholera toxin B injection. FIG. 15Bshows the packed beads after injection of 100 nM cholera toxin B.Existence of both green and red signals in the micrographs confirms thespecific binding of cholera toxin B to the GM1. After preconcentrationsurfactant elution was used for removal of receptor and receptor-toxincomplex and lipid bilayer assemblies from the biomimetic beads forsubsequent electrokinetic analysis (FIG. 15C). We have demonstrated thatsurfactant elution method is an easy and efficient method for achievingreceptor and receptor/toxin injection into the separation channel. Wehave examined the efficacy of several different ionic and nonionicsurfactants (e.g., TRITON-X-100® surfactant, TWEEN-20® surfactant,sodium dodecyl sulfate) in elution, separation and detection of GM1 andGM1-cholera toxin complex. Among the surfactants and concentrationsstudied, we concluded that 10 wt % sodium dodecyl sulfate is best suitedfor detection of the GM1-cholera toxin system. FIG. 16 shows detectionof GM1 and cholera toxin B by elution with 10 wt % sodium dodecylsulfate after preconcentration of 100 nM cholera toxin B on GM1 bearingaffinity beads. The solid line represents the elution of GM1 (Bodipy FLfluorescence) while dashed line (Alexa 555 fluorescence) represents theelution of cholera toxin B. The coincidence of the peaks for the BodipyFL and Alexa 555 dyes indicates that the eluted GM1 and cholera toxin Bare co-migrating in this system. This suggested that monitoring of theGM1 fluorescence alone can be used to detect unlabeled cholera toxin andother toxins (vide infra).

Receptor and Receptor-Toxin Separation by Miceller MicrocolumnElectrophoresis.

We studied the elution of GM1-cholera toxin B after injecting 10 μL ofaqueous solution of toxin at different concentrations. FIG. 17 shows theelectrokinetic elution of GM1 at various cholera toxin B injectionconcentrations. In each case 10 wt % sodium dodecyl sulfate was used forelution and 200V was applied over 2 cm long channel. The detector wasplaced 5 mm from start of bead microcolumn. The microchannel was 200 μmwide and 100 μm deep. Tris-glycine buffer (pH 8.4) was used as anelution buffer. The elution time of GM1 for 0 nM cholera toxin B (notoxin present in sample) was 74 seconds. As the concentration of choleratoxin B in the sample increases from 0 to 1 μM, the elution time of GM1increases from 74 to 121 seconds. FIG. 18 shows the dose-response curvefor this system that suggests that this methodology can be used todetect nanomolar concentrations of toxin in 10 μL samples. Thiscorresponds to a detection limit approaching 1 femptomole in thisun-optimized system. At 10 wt %, sodium dodecyl sulfate is well aboveits critical micelle concentration in water. During elution, GM1 andGM1/cholera toxin B complex interact with sodium dodecyl sulfatemicelles through hydrophobic interactions. We observed thatelectrophoresis dominates over electroosmosis for GM1 and GM1-choleratoxin B after their interaction with sodium dodecyl sulfate micelles. Assodium dodecyl sulfate is an anionic surfactant, its micelles are highlynegatively charged. GM1 is a 1.5 kDa receptor while cholera toxin Bconsists of 10.5 kDa subunits. These results suggest that theelectrophoretic velocity of micelles incorporating GM1 and cholera toxinis progressively decreased, as more cholera toxin is present. In theabsence of cholera toxin, GM1 containing micelles have the highestelectrophoretic velocity (and shortest elution time). Importantly, thesephenomena result in the capability to quantify cholera toxinconcentration by measuring elution times (as opposed to peak areas) andthus to the ability to measure the concentration of unlabeled choleratoxin by measuring the elution time of co-eluted fluorescently labeledGM1.

Detection of Cholera Toxin B in Complex Aqueous Samples.

To demonstrate the superb specificity of this detection strategy, wemixed cholera toxin B into water from a variety of sources includingwater from the duck pond at the University of New Mexico. 10 μL of 1 μMcholera toxin B was diluted with duck pond water to 100 nM. FIG. 19shows the electrokinetic elution of GM1 for 100 nM cholera toxin Binjection from duck pond water. Results show that specific detection ofcholera toxin B is achievable using preconcentration, elution andelectrokinetic separation of GM1 and GM1-cholera toxin B in even complexnatural aqueous samples.

It should be understood that while much of the description is related tothe use of the sensing device and methods described herein for thedetection of the presence of CBW threats, the sensing device and methodsas described are suitable for use for detection of any suitable ligand.Moreover, as stated, the device may be a useful platform to studynanofluidic interactions.

All patents and publications referenced or mentioned herein areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each such referenced patent or publicationis hereby incorporated by reference to the same extent as if it had beenincorporated by reference in its entirety individually or set forthherein in its entirety. Applicants reserve the right to physicallyincorporate into this specification any and all materials andinformation from any such cited patents or publications.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and that they are not necessarily restricted to theorders of steps indicated herein or in the claims. As used herein and inthe appended claims, the singular forms “a,” “an,” and “the” includeplural reference unless the context clearly dictates otherwise. Thus,for example, a reference to “a host cell” includes a plurality (forexample, a culture or population) of such host cells, and so forth.Under no circumstances may the patent be interpreted to be limited tothe examples or embodiments or methods specifically disclosed herein.Under no circumstances may the patent be interpreted to be limited byany statement made by any Examiner or any other official or employee ofthe Patent and Trademark Office unless such statement is specificallyand without qualification or reservation expressly adopted in aresponsive writing by Applicants.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

1. A device for analyzing a fluid sample for the presence of a target,the device comprising: an injection port; a reaction region fluidlycommunicating with the injection port via a microfluidic channel; atarget concentration mechanism confined within the reaction region, thetarget concentration mechanism being configured to releasably immobilizea labeled receptor known to bind one or more classes of targets;wherein, under a first condition, the receptor is immobilized within thereaction region; and under a second condition, the receptor is releasedby the target concentrating mechanism and free to travel away from thereaction region; a detection region fluidly communicating with thereaction region via a microfluidic channel; a fluid manipulation sourceconfigured to control movement of the fluid sample from the injectionport, through the reaction region and to the detection region; and adetector in communication with the detection region configured todifferentiate between bound receptor and unbound receptor based on thetiming of when the bound and unbound receptors move through thedetection region.
 2. The device of claim 1 wherein the targetconcentration mechanism comprises receptors immobilized in a lipidbilayer formed around a bead.
 3. The device of claim 2 wherein thesecond condition is the introduction of a surfactant.
 4. The device ofclaim 3 wherein the surfactant is sodiumdodecylsulfate.
 5. The device ofclaim 1 wherein the target concentration mechanism comprises beadsformed from a stimuli-responsive material decorated with reversiblyabsorbed receptors.
 6. The device of claim 5 wherein the secondcondition is an alteration of an environmental condition.
 7. The deviceof claim 6 wherein the alteration of an environmental condition is atemperature change.
 8. The device of claim 5 wherein thestimuli-responsive polymer is poly(N-isopopyl acrylamide).
 9. The deviceof claim 5 where the stimuli-responsive material comprises elastin-likepolypeptides.
 10. The device of claim 1 wherein the fluid manipulationsource comprises at least one power source configured to encouragemovement of the fluid in a particular direction.
 11. The device of claim1 further comprising a control lane communicating with the fluidmanipulation source, the control lane comprising: a reaction regionfluidly communicating with the injection port via a microfluidicchannel; a target concentration mechanism confined within the reactionregion, the target concentration mechanism being configured toreleasably immobilize a receptor known to bind one or more classes oftargets; wherein, under a first condition, the receptor is immobilizedwithin the reaction region; and under a second condition, the receptoris released by the target concentrating mechanism and free to travelaway from the reaction region; a detection region fluidly communicatingwith the reaction region via a microfluidic channel; and wherein sampleinjected into the lane of claim 1 is not able to enter the control lane.12. The device of claim 1 wherein the receptors are selected from thegroup consisting of: known receptors which bind one or more agents to bedetected; modified versions of receptors that bind the one or moreagents; and biomimetics of receptors that bind the one or more agents.13. The device of claim 12 wherein the receptor has been modified toenable the detection mechanism.
 14. The device of claim 13 wherein thereceptor has been modified to comprise a fluorescent label.
 15. Thedevice of claim 1 wherein the detection region includes nanochannels.16. The device of claim 1 wherein the receptor is GM1 Gangliosidemodified with a fluorescent label.
 17. A device comprising: a fluidinlet; a reaction region in fluidic communication with the fluid inletvia a nanochannel, the reaction region comprising releasable receptorsimmobilized within the reaction region; a detection region comprisingnanochannels, wherein the detection region is in fluidic communicationwith the reaction region via a nanochannel; a fluid manipulation sourceconfigured to control movement of the fluid sample through thenanochannels in the detection region; and a detector in communicationwith the detection region configured to differentiate between boundreceptor and unbound receptor based on the timing of when the bound andunbound receptors move through the detection region.
 18. The device ofclaim 17 wherein the releasable receptors are immobilized to beadspacked in the reaction region.
 19. The device of claim 18 wherein thereceptors are immobilized in a lipid bilayer formed around a bead. 20.The device of claim 18 wherein the receptors are reversibly absorbed insmart beads.
 21. The device of claim 17 comprising: a second reactionregion comprising releasable receptors immobilized with the reactionregion; and a second detection region in fluidic communication with thesecond reaction region via a second nanochannel; wherein targetintroduced into the first reaction region is prevented from entering thesecond reaction region, and second detection region, and secondnanochannel.